Muscular dystrophy is a generic name for genetic muscular diseases that show the gradual progression of muscle atrophy or weakness resulting from the repeated cycles of the breakdown or degeneration (necrosis) and regeneration of muscle fibers. Among these diseases, progressive muscular dystrophy is well known. Duchenne muscular dystrophy (DMD), one type of progressive muscular dystrophy, is the most common form of the muscular dystrophy and is caused by mutations in the dystrophin gene, which is located on the X chromosome (see non-patent document 1). Reportedly, DMD patients typically die of heart failure or respiratory failure in their 20s due to progressive muscle atrophy that began in early childhood.
The dystrophin protein (hereinafter, also simply referred to as “dystrophin”), which is located at the cytoplasmic face of the sarcolemma, plays a role in, for example, structurally maintaining muscle cells by conveying mechanical energy generated by actin-myosin-driven muscle contraction to the sarcolemma, its surrounding connective tissues, tendons, etc., in a balanced manner, and regulatorily preventing excessive impact from being applied thereto. In the case of DMD patients, their muscle fibers contain no or only scarce dystrophin protein due to mutations in the dystrophin genes. The sarcolemma is thus disrupted as a result of muscle contraction, leading to the entrance of a larger-than-normal amount of calcium ions into the muscle fibers. Excessive calcium activates enzymes, such as calpains or proteases, which break down the muscle or induce apoptosis. As a result, fibroblasts are activated to form fibrotic scar tissues, which in turn impede the regeneration of muscle cells and accelerate muscle atrophy.
Becker muscular dystrophy (BMD), another type of progressive muscular dystrophy, is also caused by mutations in the dystrophin gene, but typically affects only adults. Its symptoms also progress more slowly than DMD. DMD and BMD differ in the severity of symptoms and the rate of progression thereof, though these diseases are both caused by mutations in the dystrophin gene. This difference between DMD and BMD is explained by the reading frame rule. A premature termination codon (PTC) mutation (nonsense mutation) in dystrophin mRNA usually brings about a grave DMD phenotype (Duchenne type), whereas a mutation that does not alter the original reading frame of dystrophin mRNA (in-frame mutation) results in a milder BMD phenotype (Becker type) (see non-patent document 2). Unexpectedly, in spite of the fact that some mild BMD patients have a nonsense mutation in their dystrophin genes, the skipping of an exon comprising this nonsense mutation reportedly yields new in-frame dystrophin mRNA (see non-patent documents 3 to 6). Dystrophin (truncated dystrophin) encoded by this dystrophin mRNA lacking some such exons by skipping is shorter than normal dystrophin, but still has, to some extent, the function of structurally maintaining muscle cells. The resulting muscular dystrophy exhibits relatively mild symptoms. Also, muscle atrophy progresses at a relatively slow rate.
The fundamental therapy of muscular dystrophy remains to be established. Only symptomatic treatment for heart failure or respiratory disorder has previously been practiced, in addition to functional training or stretch to prevent joint contracture. Novel therapy for DMD, however, has been developed by the present inventors or other researchers in recent years and has encouraged expectations. This therapy is a method involving inducing exon skipping using an antisense oligonucleotide (AON) against dystrophin mRNA to convert a DMD phenotype to a BMD phenotype so that symptoms are relieved (non-patent document 3). Some different types of AONs were designed against any of splice sites or splicing-enhancing elements in order to induce exon skipping in the cells of DMD patients. These AONs successfully repaired the reading frame of dystrophin mRNA. For example, AON against an exonic splicing enhancer (ESE) in exon 19 caused the skipping of exon 19 in the cells of the DMD patients to observably form truncated dystrophin (see non-patent documents 3 to 5). Alternatively, AON against exon 51 is also often used for such patient-derived cells. These AONs are currently at the stage of clinical study (see non-patent documents 6 to 8).
Unfortunately, such AONs must be injected intramuscularly or intravenously on a regular basis. This becomes undesirable burdensome to patients. Also, the preparation of AONs in large amounts disadvantageously costs a great deal of money. In addition, when mdx mice (muscular dystrophy model mice) were treated with AON, dystrophin expression made a recovery to some extent in the skeletal muscles, but was difficult to recover in the hearts (non-patent document 9). Thus, low molecules that regulate exon skipping have been strongly demanded from a clinical standpoint. According to the reports, PTC124 (registered trademark) (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid), a low-molecular-weight non-aminoglycoside nonsense mutation suppressor compound, is capable of treating some DMD patients having a nonsense mutation (non-patent documents 10 and 11) and is currently under phase IIb clinical trial in the USA, etc. PTC124 is a therapeutic drug that induces ribosome to read through a premature termination codon (PTC) during translation so that the expression of full-length functional dystrophin is recovered to some extent. Its influence on the nonsense mutation-dependent mRNA decay mechanisms of the other genes is still uncertain.
The present inventors have identified a Cdc-like kinase (Clk)-specific kinase inhibitor TG003 (compound represented by the general formula (1) described later wherein R1 and R2 each represent a methyl group; and R3 represents a methoxy group). The compound TG003 influences splicing both in vitro and in vivo (see non-patent documents 12 and 13). The patent application (see patent document 1) in which the present inventors are involved discloses that TG003 has the effect of regulating alternative splicing mediated by the phosphorylation of SR protein and that diseases such as cancer may be prevented or treated using this effect. It has been unknown so far that TG003 is capable of promoting the skipping of an exon in the dystrophin gene.